Personalized methods are urgently needed for monitoring tumor status and assessing recurrence or treatment failure. Conventional means of monitoring include clinical signs, symptoms, laboratory results, and expensive tests such as radiographic imaging (CT scan, bone scan, PET scan). However, more sensitive and, ideally, more quantitative methods would be advantageous. We seek to combine two powerful molecular technologies to monitor tumors in blood by identifying and tracking, a "cancer mutation signature" specific for each individual patient. Four core observations underlie our goal of developing such a test. (1) In several recent studies, the Vogelstein group demonstrated that 100-200 somatic mutations generally occur in the exome of breast, colon, and other cancers. (2) Recent technological advances in massively parallel sequencing allow a vast amount of sequencing over an entire genome (or subset thereof) and would permit the detection of somatic tumor mutations in multiple cancer-related genes within a relatively short time. (3) Previous studies have shown that cancer-specific DNA released from necrotic or apoptotic cancer cells can be detected in plasma. (4) Technical advances in Pyrophosphorolysis-Activated Polymerization (PAP), a highly sensitive and specific method developed by the founder of this company, enables detection of a single copy of DNA harboring such cancer- specific signatures in both the plasma and cellular compartments of blood. The proposed study will develop operational criteria for a "cancer mutation signature" by analyzing tumor and normal DNA, as well as serial blood samples from four breast cancer patients. [What makes this project unique is the fact that we will identify a set of tumor mutations for each patient that (1) specifically defines the individual tumor of that patient and (2) are not present in the patient's matched normal tissue, providing a truly personalized "cancer signature".] Using massively parallel sequencing, about 20 mutations will be detected in the tumor DNA of each patient, confirmed by capillary sequencing, and shown to be absent in the normal DNA. PAP assays will be developed for each of five mutations chosen per patient. Criteria for a reliable "cancer mutation signature" will be determined. It is hypothesized that between two and five somatic mutations will be required per cancer, given the possibility of confounders. With the "cancer mutation signature", blood samples will be tested to monitor the patient at diagnosis and during the subsequent clinical course. Outcomes will be compared to the standard patient monitoring modalities. Success in this Phase I feasibility study will lead to a Phase II clinical study and eventually to a commercial test for individualized personal testing for cancer patients. PUBLIC HEALTH RELEVANCE: For patients with cancer, better methods for monitoring therapy or recurrence could improve outcome while reducing cost. Conventional means of monitoring cancer progression are not sensitive enough to determine the therapeutic effectiveness or to detect recurrence of the tumor in a timely manner. With the power of the revolutionary next-generation sequencing, it becomes possible to identify an individual's unique cancer mutation signature (a method applicable to all cancer types), in which single molecules of the cancer signature can be detected in blood. The monitoring of therapy or recurrence (MOTOR) with a combination of massively parallel sequencing and PAP (Pyrophosphorolysis-Activated Polymerization) has the potential to revolutionize cancer treatment.